Human Tdp1 Cleaves a Broad Spectrum of Substrates, Including Phosphoamide Linkages

Abstract

Human tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes the phosphodiester bond between a DNA 3 end and a tyrosyl moiety. In eukaryotic cells, this type of linkage is found in stalled topoisomerase I-DNA covalent complexes, and Tdp1 has been implicated in the repair of such complexes in vivo. We confirm here that the Tdp1 catalytic cycle involves a covalent reaction intermediate in which a histidine residue is connected to a DNA 3-phosphate through a phosphoamide linkage. Most surprisingly, this linkage can be hydrolyzed by Tdp1, and unlike a topoisomerase I-DNA complex, which requires modification to be an efficient substrate for Tdp1, the native form of Tdp1 can be removed from the DNA. The spinocerebellar ataxia with axonal neuropathy neurodegenerative disease is caused by the H493R mutant form of Tdp1, which shows reduced enzymatic activity and accumulates the Tdp1-DNA covalent intermediate. The ability of wild type Tdp1 to remove the stalled mutant protein from the DNA likely explains the recessive nature of spinocerebellar ataxia with axonal neuropathy. In addition to its activity on phosphotyrosine and phosphohistidine substrates, Tdp1 also possesses a limited DNA and RNA 3-exonuclease activity in which a single nucleoside is removed from the 3-hydroxyl end of the substrate. Furthermore, Tdp1 also removes a 3 abasic site and an artificial 3-biotin adduct from the DNA. In combination with earlier data showing that Tdp1 can use 3-phosphoglycolate as a substrate, these data suggest that Tdp1 may function to remove a variety of 3 adducts from DNA during DNA repair.Tyrosyl-DNA phosphodiesterase (Tdp1) 2 was discovered as an enzymatic activity from Saccharomyces cerevisiae that specifically hydrolyzes the phosphodiester linkage between the O-4 atom of a tyrosine and a DNA 3Ј-phosphate (1). This type of linkage is typical of the covalent reaction intermediate produced when a type IB topoisomerase cleaves one strand of DNA. Type IB topoisomerases are ubiquitous enzymes that perform essential functions in key cellular processes such as replication, recombination, and transcription (2-6). Following cleavage and DNA relaxation, the topoisomerase normally religates the DNA strand and dissociates from the DNA (4). After cleavage near certain DNA lesions or modified nucleotides, or in the presence of the anticancer drug camptothecin, religation is blocked, and the topoisomerase becomes covalently trapped on the 3Ј end of the cleaved DNA strand (7-10). At the time of the discovery of Tdp1, Yang et al.(1) suggested that the enzyme might be involved in the removal of such covalently stalled topoisomerase I molecules from the DNA. At present, extensive genetic, biochemical, and cell biological data support the view that Tdp1 is involved in the repair of topoisomerase I-DNA covalent lesions in eukaryotic cells (11)(12)(13)(14)(15)(16).Tdp1 is a member of the phospholipase D (PLD) superfamily, which is characterized by two "HKD" motifs that provide the conserved active site residues (17). The diverse members of this family ful...

Spermiogenesis and DNA Repair: A Possible Etiology of Human Infertility and Genetic Disorders

“…Using confocal microscopy, we recently confirmed the presence of topoisomerase IIb (TOP2B, topoIIb) distributed in foci during steps 9 to 13 of mouse spermiogenesis but did not observe the presence of the a isoform at these steps [Leduc et al unpublished results]. Interestingly, we identified the presence of tyrosyl-DNA phosphodiesterase 1(TDP1), an enzyme known to resolve topoisomerasesmediated DNA damage [Interthal et al 2005;Nitiss et al 2006;Raymond and Burgin 2006]. The presence of TDP1 is coincident with the appearance of topoIIb in ES.…”

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

“…In addition, wild-type Tdp1 has been shown to hydrolyze the covalent phosphoamide intermediate proposed to form in SCAN1 cells between the mutant Tdp1 and the DNA (Fig. 3E) [52]. Other naturally occurring substrates for Tdp1 include 3′-abasic sites, 3′-nucleosides, and 3′-ribonucleosides (Fig.…”

“…Other naturally occurring substrates for Tdp1 include 3′-abasic sites, 3′-nucleosides, and 3′-ribonucleosides (Fig. 4C) [52]. However, it is interesting to note that the Tdp1 is only able to remove a single terminal 3′-mononucleoside and is incapable of processing a DNA end containing a 3′-phosphate [52], which demonstrates that Tdp1 cannot act as a 3′-exonuclease.…”

“…4C) [52]. However, it is interesting to note that the Tdp1 is only able to remove a single terminal 3′-mononucleoside and is incapable of processing a DNA end containing a 3′-phosphate [52], which demonstrates that Tdp1 cannot act as a 3′-exonuclease. Yeast Tdp1 has also been shown to process a topoisomerase II (Top2) cleavage complex or a 5′-phosphotyrosyl bond [53].…”

“…Specifically, phosphotyrosine analogues, such as 3′-(4-nitro)phenol and 3′(4-methyl)phenol, have been used in kinetic analysis of the Tdp1 catalytic mechanism [32]. Other artificial substrates include a 3′-linker-biotin [52] and a fluorescent 3′-4-methylumbelliferone [55], which could potentially be employed in high-throughput screens for Tdp1 inhibitors, similar to how the 3′-BV tag has been used (discussed below).…”